Partially demineralized cortical bone constructs

ABSTRACT

The invention is directed toward a sterile bone structure for application to a bone defect site to promote new bone growth at the site comprising a partially demineralized cortical bone structure, said bone structure comprising a cross sectional surface are ranging from 85% to 95% of the original bone surface area before demineralization with the remaining partially demineralized cortical bone structure having an outer demineralized layer ranging in thickness from about 0.05 mm to about 0.14 mm and a mineralized core.

RELATED APPLICATION

The present invention is a continuation-in-part of U.S. patentapplication Ser. No. 09/677,891 filed Oct. 3, 2000.

FIELD OF INVENTION

The present invention is generally directed toward a surgical boneproduct and more specifically is a shaped partially demineralizedallograft bone device or construct with a mineralized central section.

BACKGROUND OF THE INVENTION

The use of substitute bone tissue dates back around 1800. Since thattime research efforts have been undertaken toward the use of materialswhich are close to bone in composition to facilitate integration of bonegrafts. Development have taken place in the use of grafts of a mineralnature such as corals, hydroxyapatites, ceramics or synthetic materialssuch as biodegradable polymer materials. Surgical implants should bedesigned to be biocompatible in order to successfully perform theirintended function. Biocompatibility may be defined as the characteristicof an implant acting in such a way as to allow its therapeutic functionto be manifested without secondary adverse affects such as toxicity,foreign body reaction or cellular disruption.

Human allograft tissue is widely used in orthopaedic, neuro-,maxiliofacial, podiatric and dental surgery. The tissue is valuablebecause it is strong, biointegrates in time with the recipient patient'stissue and can be shaped either by the surgeon to fit the specificsurgical defect or shaped commercially in a manufacturing environment.Contrasted to most synthetic absorbable or nonabsorbable polymers ormetals, allograft tissue is bioinert and integrates with the surroundingtissues. Allograft bone occurs in two basic forns; cancellous andcortical. Cortical bone is a highly dense structure comprised of triplehelix strands of collagen fiber, reinforced with hydroxyapatite. Thecortical bone is a compound structure and is the load bearing componentof long bones in the human body. The hydroxyapatite component isresponsible for the high compressive strength of the bone while thecollagen fiber component contributes in part to torsional and tensilestrength.

Many devices of varying shapes and forms can be fabricated fromallograft cortical tissue by machining and surgical implants such aspins, rods, screws, anchors, plates, intervertebral spacers and the likehave been made and used successfully in human surgery. These engineeredshapes are used by the surgeon in surgery to restore defects in bone tothe bone's original anatomical shape. This treatment is well known inthe art and is commercially available as demineralized bone.

Allograft bone is a logical substitute for autologous bone. It isreadily available and precludes the surgical complications and patientmorbidity associated with obtaining autologous bone as noted above.Allograft bone is essentially a collagen fiber reinforced hydroxyapatitematrix containing active bone morphogenic proteins (BMP) and can beprovided in a sterile form. The demineralized form of allograft bone isnaturally both osteoinductive and osteoconductive. The demineralizedallograft bone tissue is fully incorporated in the patient's tissue by awell established biological mechanism. It has been used for many yearsin bone surgery to fill the osseous defects previously discussed.

Demineralized allograft bone is usually available in a lyophilized orfreeze dried and sterile form to provide for extended shelf life. Thebone in this form is usually very coarse and dry and is difficult tomanipulate by the surgeon. One solution to use such freeze dried bonehas been provided in the form of a commercially available product,GRAFTON®, a registered trademark of Osteotech Inc., which is a simplemixture of glycerol and lyophilized, demineralized bone powder of aparticle size in the range of 0.1 cm to 1.2 cm as is disclosed in U.S.Pat. No. 5,073,373 issued Dec. 17, 1991 forming a gel. Similarly U.S.Pat. No. 5,290,558 issued Mar. 1, 1994, discloses a flowabledemineralized bone powder composition using a osteogenic bone powderwith large particle size ranging from about 0.1 to about 1.2 cm. mixedwith a low molecular weight polyhydroxy carrier possessing from 2 toabout 18 carbons comprising a number of classes of different compoundssuch as monosaccharides, disaccharides, water dispersibleoligosaccharides and polysaccharides.

A recent version of GRAFTON® product uses relatively large demineralizedparticles in the carrier to create a heterogenous mixture which providesbody or substance to the composition. This material is useful in fillinglarger defects where some degree of displacement resistance is needed bythe filler.

The advantages of using the bone particle sizes as disclosed in the U.S.Pat. Nos. 5,073,373 and 5,290,558 patents previously discussed werecompromised by using bone lamellae in the shape of threads or filamentshaving a median length to median thickness ratio of about 10:1 andhigher while still retaining the low molecular weight glycerol carrier.This later prior art is disclosed in U.S. Pat. Nos. 5,314,476 issued May24, 1994 and U.S. Pat. No.5,507,813 issued Apr. 16, 1996 and the tissueforms described in these patents are known commercially as the GRAFTON®Putty and Flex, respective.

The combination of natural cortical bone with very desirable mechanicalstrength and the addition of synthetic (recombinant) BMPs provides asuperior form of tissue for surgical use retaining all of the mechanicalproperties of the cortical component and the accelerated healing offeredby the BMP's.

U.S. Pat. No. 5,972,368 issued on Oct. 26, 1999 discloses the use ofcortical contructs (e.g. a cortical dowel for spinal fusion) which arecleaned to remove all of the cellular material, fat, free collagen andnon-collagenous protein leaving structural or bound collagen which isassociated with bone mineral to form the trabecular struts of bone. Itis stated that the natural crystalline structure of bone is maintainedwithout the risk of disease transmission or significant immunogenicity.Thus the shaped bone is processed to remove associated non-collagenousbone proteins while maintaining native bound collagen materials andnaturally associated bone minerals. Recombinant BMP-2 is then drippedonto the dowel surface. It could also be added to the cortical bone bysoaking in the BMP-2 solution. As noted, this reference teaches theremoval of all non-collagenous bone proteins which necessarily includeall the naturally occurring BMP's and relies upon the addition ofrecombinant BMP-2 in a specific and empirically determinedconcentration. The naturally occurring BMP's are present in aconcentration unique for each specific BMP protein and has beenoptimized by nature. The '368 patent teaches complete removal of thenatural BMP's by demineralization and relies solely on the addedrhBMP's. The surface of a machined cortical bone surface ischaracterized by a wide variety of openings resulting from exposure bythe machining process of the Haversian canals present throughoutcortical bone. These canals serve to transport fluids throughout thebone to facilitate the biochemical processes occurring within the bone.They occur at variable angles and depths within the bone. Hence, whenthe machining occurs, the opening will be varied and unpredictableresulting in a highly variable and uncontrolled amount of BMP enteringthe surface of the bone.

In WO99/39,757 published Aug. 12, 1999, an osteoimplant is disclosedwhich uses partially demineralized bone elements and adjacentsurface-exposed collagen to form chemical linkages to bond the elementsinto a solid aggregate. It is noted in the Description of the PreferredEmbodiments, that ‘when prepared from bone derived elements that are“only superficially demineralized” that the osteoimplant will possess afairly high compression strength approaching that of natural bone. FIG.2 illustrates bone-derived stacked sheets having a fully or partiallydemineralized outer surface 21 with surface exposed collagen and anondemineralized or partially demineralized core 22. As noted in Example1, the bone sheets approximately 1.5 mm thick were placed in a 0.6N HClsolution for 1.5 hours with constant stirring, washed in water for 5minutes and soaked for 1.5 hours in phosphate buffered saline. InExample 3 the bone-derived sheets from cortical bone were treated for 10minutes in 0.6N HCl to expose surface collagen. Bone cubes derived fromhuman cancerous bone were treated to expose surface collagen at theouter borders of the cube. In Example 4, human cortical bone-derivedsheets approximately 1 mm thick were surface demineralized for 15minutes in 0.6N HCl and in Example 5, human cortical bone derived sheetsapproximately 2 mm thick were surface demineralized for 1 hour in 06NHCl.

U.S. Pat. No. 5,899,939, issued May, 1999, to the same inventor as theforeign patent noted in the paragraph above, discloses a bone derivedimplant made up of one or more layers of fully mineralized or partiallydemineralized cortical bone, and optionally one or more layers of someother material. The layers of the implant are assembled into a unitarystructure to provide an implant.

In U.S. Pat. No. 5,861,167, issued Jan. 19, 1999, a toothroot is shownto have selective parts of the surface removed by acid to improvesubsequent attachment of the tooth in conjunction with periodontalsurgery. Similarly U.S. Pat. No. 5,455,041 utilized treatment bydemineralizing the tooth root surface with citric acid applied for oneminute to effect reattachment of collagen fibers to the root surface andadding growth factors onto the surface of the demineralized root.

Partial demineralization of bone is also disclosed in the Journal ofSurgical Research Vol. 59, pages 614-620 (1995) in the articleSterilization of Partially Demineralized Bone Matrix: The Effects ofDifferent Sterilization Techniques on Osteogenetic Properties whereparticles of bone of 500 microns were treated for 24 hours at 4 degreesC. with 0.6 N HCl with the extent of decalcification determined to be20% and placed in the bone site. New bone formation was noted after thepassage of six weeks.

In French Patent Applications Numbers 2,582,517 and 2,582,518 treatmentof fragments of bones taken from animals, primarily cattle werepartially demineralized and tanned with glutaraldehyde. The boneelements to be implanted are cut to the desired shape from an ox bonewhich has been subjected to a treatment comprising a degreasing stepwith an organic solvent such as ethanol, a demineralization step with acalcium extraction agent such as hydrochloric acid and tanning withglutaraldehyde and subsequent washings. Similar demineralization of boneis shown in U.S. Pat. No. 5,585,116 issued Dec. 17, 1996. This patentalso notes that it is known that partial demineralization facilitatesintegration of a bone graft. This is accordingly followed by differentcomplementary steps which are intended either to deproteinize the bonecompletely or to act on the nature of the proteins which then remainlinked within the bone matrix or else to increase this proportion ofproteins.

It is desirable to make the surface of the bone more conductive toreceiving BMP's and other additives without losing the desirable highmechanical strength properties of the cortical bone. It is alsodesirable to leave most of the naturally occurring protein intact in thebone in such a way as to expose just enough of the bone surface to freethe natural BMP's present on the surface. Since demineralization alsoreduces the cross sectional area of the bone construct, the boneconstruct must retain its shape and structural integrity.

Accordingly, the prior art only partially addresses the problemsinherent in correcting surgical defects.

SUMMARY OF THE INVENTION

The present invention is directed toward the treatment of the surface ofcortical bone constructs to modify the surface by removing a layer ofthe inorganic mineral hydroxyapatite material leaving the mechanicalproperties of the bone constructs substantially unchanged whileproviding a surface that allows the addition of BMP's and otherdesirable additives to be introduced to the surface and thereby enhancethe healing rate of the cortical bone in surgical procedures.

The subject formulation is a demineralized bone structure forapplication to a bone defect site to promote new bone growth at the sitecomprising a partially demineralized cortical bone structure, said bonestructure comprising a cross sectional surface are ranging from 85% to95% of the original bone surface area before demineralization with theremaining partially demineralized cortical bone structure comprising anouter demineralized layer ranging in thickness from about 0.05% to about0.14%. The structure is designed to present the bone matrix and ademineralized surface layer for reception of bone morphogenetic proteins(BMP) and other desired additives. The macrostructure of the highlyporous demineralized surface layer serves both as an osteoconductivematrix and to signal the patient's tissue and cells to initiate thegrowth of new bone (osteoinduction).

It can be seen that the prior art has attempted to replicate to somedegree the present invention by flash demineralization of the surface orfull demineralization of the structure.

It is thus an object of the invention to provide a shaped bone implantconstruct having a partially demineralized cortical bone layer with aninterior mineralized bone section to provide compression strength to theimplant bone construct.

It is an object of the invention to utilize a partially demineralizedshaped bone implant structure to approximate the mechanical strengthcharacteristics of natural bone to provide overall strength and initialdurability to the structure.

It is yet another object of the invention to provide a partiallydemineralized shaped bone implant structure to provide a strong implantstructure of a predetermined shape and size for implantation.

It is also an object of the invention to provide a bone derivedstructure which can effective hold medical and biological compositionwhich promote new bone growth and accelerate healing.

It is an additional object of the invention to use a BMP additive in thedemineralized layer of the bone structure.

It is an still additional object of the invention to use a solublesilver additive in the demineralized layer of the bone structure.

It is also an object of the invention to create a bone structure whichcan be easily handled by the physician.

These and other objects, advantages, and novel features of the presentinvention will become apparent when considered with the teachingscontained in the detailed disclosure which along with the accompanyingdrawings constitute a part of this specification and illustrateembodiments of the invention which together with the description serveto explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a partially demineralized rod or dowelaccording to the invention;

FIG. 2 is a perspective view of a partially demineralized screwaccording to the invention;

FIG. 3 is a perspective view of a partially demineralized anchoraccording to the invention;

FIG. 4 is a perspective view of a partially demineralized wedgeaccording to the invention;

FIG. 5 is a perspective view of a partially demineralized fusion ringaccording to the invention;

FIG. 6 is a perspective view of a partially demineralized compositestructure according to the invention;

FIG. 7 is a photograph of a 35× enlarged cross sectional view of apartially demineralized rod treated with 0.6N HCl for 30 minutes;

FIG. 8 is a photograph of a 35× enlarged cross sectional view of apartially demineralized rod treated with 0.6N HCl for 60 minutes;

FIG. 9 is a photograph of a 35× enlarged cross sectional view of apartially demineralized rod treated with 0.6N HCl for 90 minutes;

FIG. 10 is a photograph of a 35× enlarged cross sectional view of apartially demineralized rod treated with 0.6N HCl for 120 minutes;

FIG. 11 is a photograph of a 35× enlarged cross sectional view of apartially demineralized rod treated with 0.6N HCl for 180 minutes;

FIG. 12 is a graph showing bending displacement in relation to acid soaktime; and

FIG. 13 is a graph showing weight loss during partial demineralizationin relation to acid soak time.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed towards a treated partiallydemineralized cortical bone construct which can be placed in a bonedefect area to heal bone defects. The term cortical bone construct meansany shaped bone device such as rods, pins, dowels, screws, plates,wedges, fusion rings, intervertaebral spacers and composite assemblies.The aforementioned listing is exemplary only and is not to construed asrestrictive.

The preferred embodiment and the best mode as shown in FIGS. 1 and 7-11and shows a cylindrical cortical bone construct 10 with its surface 12modified by acid treatment to remove a layer of the inorganic, mineral,hydroxyapatite bone material in such a way as to leave the mechanicalproperties substantially unchanged. While the bone material is referredto as hydroxyapatite in this application, in actuality the chemistry andstructure of natural bone mineral is different as natural bone mineralcontains carbonate ions, magnesium, sodium, hydrogen phosphate ions andtrace elements and a different crystalline structure thanhydroxyapatite.

The unique features of bone that makes it desirable as a surgicalmaterial are, its ability to slowly resorb and be integrated into thespace it occupies while allowing the bodies own healing mechanism torestore the repairing bone to its natural shape and function by amechanism known in the art as creeping substitution. The second featureis the high mechanical strength arising from the collagen fiberreinforced hydroxyapatite compound structure. The creeping substitutionmechanism, takes considerable time and some forms of cortical bone intheir natural, unmodified biological state have been found to persistfor over one year before completely remodeling. Thus a means ofaccelerating the rate of biointegration of cortical bone would improvethe rate of healing and benefit the recipient patient.

It is well known that bone contains osteoinductive elements known asbone morphogenetic proteins (BMP). These BMP's are present within thecompound structure of cortical bone and are present at a very lowconcentrations, e.g. 0.003%. Based upon the work of Marshall Urist asshown in U.S. Pat. No. 4,294,753, issued Oct. 13, 1981 the properdemineralization of cortical bone will expose the BMP and present theseosteoinductive factors to the surface of the demineralized materialrendering it significantly more osteoinductive. The removal of the bonemineral leaves exposed portions of collagen fibers allowing the additionof BMP's and other desirable additives to be introduced to thedemineralized outer treated surface of the bone structure and therebyenhances the healing rate of the cortical bone in surgical procedures.The treatment process also exposes the naturally occurring BMP's at thesurface and renders the surface with biological properties similar tofull demineralized bone (DBM). The inner mass 14 of the bone mineral ofthe shaped construct would be left intact to contain the naturallyoccurring BMP's and trace elements as noted above. Such a product wouldbe beneficial in spinal fusion, fracture fixation and similarorthopaedic and neurological procedures where rapid healing without lossof strength of implant is required. Partially demineralized rods 16 asshown in FIGS. 1 and FIGS. 7-11 will retain various degrees of stiffnessinversely proportional to the degree of demineralization and retentionof core mass. The partially demineralized rods have a demineralizedouter section 18 of exposed collagen matrix and a cortical bone core 20.

Experiments conducted by the Applicants have discovered that the surfaceof cortical bone constructs can be modified by acid treatment to removea layer of the inorganic, mineral, hydroxyapatite material in such a wayas to leave the mechanical properties substantially unchanged or toprovide a construct having suitable compression and bending strength.This then allows the addition of BMP's and other desirable additives tobe introduced to the surface and thereby enhance the healing rate of thecortical bone in surgical procedures. The process also exposes thenaturally occurring BMP's near the surface and renders the surface withbiological properties similar to fully demineralized bone (DMB). Theinner mass of the bone construct would be left intact to contain thenaturally occurring BMP's.

It was found that when allograft cortical pins of 2.0 mm diameter weretreated as noted below in Example 1; and the pins were soaked for 15 to30 minutes in a 0.6N solution of HCl that there was minimal loss ofbending strength of the rod even when the diameter of the rod wasreduced from 3 to 5% and the outer layer was demineralized. Thedemineralized layer ranged from about 0.05 to about 0.08 mm reducing themineralized portion diameter from 0.10 mm to 0.1 6mm after 15 to 30minutes of soaking in the 0.6N HCl acid bath.

EXAMPLE 1

Allograft cortical bone pins were prepared by machining femoral ortibial cortical bone. Pins were prepared with diameter of approximately2.0 mm and a length of 4 cm. The bulk bone segments from which the pinswere cut were chemically cleaned before machining by soaking:

1) 30 minutes in an aqueous antibiotic solution of Gentamycin. Thisreduces and eliminates any bioburden introduced by handling the bone.

2) 30 minutes in an aqueous detergent at 95° F. using ultrasonic energyto enhance penetration. This loosens and removes the lipid elementspresent in and on the bone.

3) 60 minutes in a 70/30%v/v ethanol/water solution. This furtherremoves any lipid elements remaining after the detergent wash in step 2,above.

4) The final cut pins were given a final soak in a fresh solution of theethanol/water cleaning solution.

5) The pins were cut in half and then immersed in a 0.6 N solution ofHydrochloric Acid (HCl). Half of each pin was immersed for varying timesand the other half was retained as an untreated control.

6) The acid treatment was done at room temperature, 23° C.

7) Acid immersion was done for 30, 60, 90, 120 and 180 minutes. The pinswere immersed in the acid solution and agitated with gentle mechanicalstirring.

8) After the appropriate elapsed time the pins were removed, washed withsterile, pure (USP Sterile) water until the wash discard was at neutralpH.

9) The pins were then lyophilized and packaged in a moisture permeablecontainer.

For purpose of this example, the above treatments were done in alaboratory setting. In a commercial process, the procedures would bedone in a sterile, clean room facility.

The acid treatment can be controlled to remove a small layer of the bonemineral layer leaving a highly porous and compressible surface layerwhile inducing no change to the inner mass of the construct. Bycontrolling the acid concentration, temperature and time of exposure, alayer up to 0.06 mm can be removed and a layer 0.08 mm demineralized andhave the cortical pin experience substantially no loss of mechanicalproperties as measured by a three-point bending test. This is anunexpected result in that mass loss should have a deleterious effect onbending resistance since the bending moment of a cylindrical beam is afunction of the third power of the diameter.

Weight Loss, % Demineralizadon Time (n = 3) [0.6 N HCl @ 23° C.] AverageStd Dev  30 minutes 31.8 3.2  60 38.1 1.9  90 48.2 1.2 120 56.1 6.4 18064.9 2.9

The thickness of the demineralized layer was also measured. For eachtreated pin, the thickness of the demineralized layer was measured sixtimes by starting at the top of the bone traveling clockwiseapproximately 60°. The following data was measured:

Thickness of Demineralized Layer Demineralization Time (mm) [0.6 N HCl @23° C.] Average (n = 6)  30 miutes 0.08  60 0.11  90 0.14 120 0.17 1800.25

The treated and control pins were subjected to a three-point bendingtest. Force-displacement calculations were made from the test results asare shown in FIG. 12. Bending displacement appears to be directlyproportional to the acid soak time after 30 minutes. It is noteworthythat the bending displacement is equivalent for the 30 minute soak timeand the untreated control. Also note that the 30 minute acid treatmentdid reduce the diameter of the pin 0.12 mm.

Scanning electron micrographs of the treated and control pins were madeand can be seen in the FIGS. 7, 8, 9, 10, and 11 reflecting photographsof the same. It can be clearly seen that the Haversian canals can beseen in the cross-section of the acid treated pins and show the removalof the mineral layer at the surface at 35×, revealing the open pores inthe demineralized layer exposed by the acid treatment.

This data demonstrates that surface demineralization can be achieved toremove significant amounts of the surface mineral layer withoutaffecting the bulk mechanical strength.

Similar treatments were done for other machined cortical shapes using0.6N HCl at 23° C. for 10 minutes:

EXAMPLE 2 Anterior Lumbar Intervertebral Fusion Ring (FRA) EXAMPLE 3Posterior Lumbar Intervertebral Fusion Block (PLIF) EXAMPLE 4 AnteriorCervical Fusion Ring (ACF) EXAMPLE 5 Allograft Bone Screw

In all these examples, the surface of the machined cortical shape wasmodified without loss of the key details and dimensions machined intothe surface.

The following shows the diameter change, the change in surfacemorphology, and the size of the demineralized layers in cylindrical pinsthat were demineralized in 0.6N HCl in 30, 60, 90, 120, and 180 minutes.

1. Diameter Change

The diameter of each pin was measured in 3 places along the pin. Themeasurements were recorded on the length of the photograph at 1.5 cm,6.5 cm, and 11.5 cm on the pin. Each measurement is recorded in thetables below. The bottom column in each “difference between the treatedand untreated pins” is the actual size difference. The pin was magnified×35 so that the measurements were each divided by 35 to arrive at theactual difference diameter change.

Pin 1 - 30 minute soak Untreated: Pin 1-B1 Left Side Middle Right SideMeasurement 6.6 cm 6.4 cm 6.5 cm Treated: Pin 1-B2 Left Side MiddleRight Side Measurement 6.0 cm 6.0 cm 6.2 cm

Difference between the treated and untreated pins

Left Side Middle Right Side Measurement  0.6 cm  0.4 cm  0.3 cm Actual0.017 cm 0.011 cm 0.009 cm Difference Average diameter change for pin 1:0.012 cm (0.12 mm)

Pin 2 - 60 minute soak Untreated: Pin 2-A2 Left Side Middle Right SideMeasurement 6.9 cm 7.1 cm 6.5 cm Treated: Pin 2-A2 Left Side MiddleRight Side Measurement 6.3 cm 6.3 cm 6.2 cm

Difference between the treated and untreated pins

Left Side Middle Right Side Measurement  0.6 cm  0.5 cm  0.3 cm Actual0.017 cm 0.023 cm 0.009 cm difference Average diameter change for pin 2:0.016 cm (0.16 mm)

Pin 3 - 90 minute soak Untreated: Pin 3-C1 Left Side Middle Right SideMeasurement 7.1 cm 7.1 cm 6.9 cm Treated: Pin 3-C2 Left Side MiddleRight Side Measurement 5.9 cm 5.6 cm 5.4 cm

Difference between the treated and untreated pins

Left Side Middle Right Side Measurement  1.2 cm  1.5 cm  1.5 cm Actual0.034 cm 0.043 cm 0.043 cm difference Average diameter change for pin 3:0.040 cm (0.40 mm)

Pin 4 - 120 miuute soak Untreated: Pin 4-A1 Left Side Middle Right SideMeasurement 6.9 cm 6.5 cm 6.6 cm Treated: Pin 4-A1 Left Side MiddleRight Side Measurement 5.1 cm 5.2 cm 4.9 cm

Difference between the treated and untreated pins

Left Side Middle Right Side Measurement  1.8 cm 1.6 1.7 Actual 0.051 cm0.046 cm 0.049 cm Difference Average diameter change for pin 4: 0.049 cm(0.49 mm)

Pin 5 - 180 minute soak Untreated: Pin 5-A2 Left Side Middle Right SideMeasurement 6.9 cm 6.9 cm 6.7 cm Treated: Pin 5-A2 Left Side MiddleRight Side Measurement 5.3 cm 4.6 cm 5.0 cm

Difference between the treated and untreated pins

Left Side Middle Right Side Measurement  1.6 cm  2.3 cm  1.3 cm Actual0.046 cm 0.066 cm 0.037 cm difference Average diameter change for pin 5:0.050 cm (0.50 mm)

2. Surface Morphology

The surfaces of the treated pins were compared to the surfaces of theuntreated pins.

Pin Number Surface Morphology 1-B1 Particles are held very tightlytogether. There are small gaps in the bone. It looks somewhat rigid.1-B2 Looks looser than 1-B1. Very rough looking. Can see looseparticles. There are many holes in the bone. Appears to have moredimension/depth than 1-B1. 2-A1 Particles are held tightly together.There are many small gaps in the bone. 2-A2 There are many looseparticles. The gaps are wider than 2-A1. 3-C1 Very dense andrigid-looking. Particles are held tightly together. 3-C2 Not as dense as3-C1. There are many small surface holes and a couple of looseparticles. 4-A1 Particles held tightly together. Surface appears veryrigid. 4-A2 Surface smoother than 4-A1. There are many surface holes(some deep enough to see the next layer some just forming). A couple ofloose particles. 5-A1 Very dense and rigid. Small gaps. 5-A2 Smootherthan 5-A1. Many surface holes. Towards the top of the slide, the boneappears bumpy. Gaps are wider than in 5-A1.

3. Thickness of the Demineralized Layer

For each treated pin, the thickness of the demineralized layer wasmeasured 6 times and the average per pin was calculated and recorded.Note: The measurements started at the top of the bone and recordedclockwise at approximately 60° intervals. (A magnifying glass with a cmruler on it was used to measure the demineralized layer of each pin).

Pin Measurement Number Average Number 1 2 3 4 5 6 Thickness 1-B2 0.09 mm0.09 mm 0.06 mm 0.11 mm 0.06 mm 0.09 mm 0.08 mm 2-A2 0.11 mm 0.09 mm0.09 mm 0.11 mm 0.14 mm 0.11 mm 0.11 mm 3-C2 0.14 mm 0.06 mm 0.03 mm0.17 mm 0.29 mm 0.14 mm 0.14 mm 4-A2 0.17 mm 0.20 mm 0.20 mm 0.17 mm0.11 mm 0.14 mm 0.17 mm 5-A2 0.26 mm 0.23 mm 0.20 mm 0.23 mm 0.29 mm0.29 mm 0.25 mm

4. Results

The length of acid soak has an effect on the diameter of the pin. Whilelonger the pin is soaked in 0.6N HCl, the more the diameter changes insize (the diameter gets smaller), a relatively constant diameter wasreached after the 120 minutes of soak in the HCC. The average diameterchange for the pin soaked for 30 minutes was 0.12 mm; for 60 minutes was0.16 mm; for 90 minutes was 0.40 mm; and for 120 minutes was 0.49 mm and180 minutes was 0.50 mm. The cross-section slides show that while thediameter of the pins decreased at an increased amount from soak minutes60 to 90 lessening from soak minutes 90 to 120, it remainingsubstantially constant thereafter. The thickness of the demineralizedlayer increased almost linearly.

The surface morphology was also affected by the acid soaks. All the pinswere viewed under a magnification of 100×. The slides of the untreatedpins looked rigid, the particles were tightly held into place making thebone to appear dense, and there were small gaps on some sections of thebones. The slides of the treated pins looked completely different thanthe untreated pins. The treated-pin slides show loose particles, surfaceholes, widened gaps, and the bones appear to be less dense.

Overall, the length of acid soak time affects the three areas tested inthis study:

1. The longer the pin soaks in 0.6N HCl, the actual diameter of the pindecreases up until 120 minutes of acid soak.

2. The longer the pin is in the acid soak, the thickness of thedemineralized layer on the bone increases and the core mineralizedportion decreases.

3. The acid also has an effect on the surface morphology of the bone. Itchanges the surface morphology from appearing very dense and rigid (whenuntreated) to having loose particles and becoming somewhat smoother(when treated).

It is valuable to add soluble silver (e.g. AgNO₃) to the surface treatedcortical bone structure. This will provide biostatic properties to theconstruct, i.e., it will inhibit any growth of microorganisms which maybe resident on the surface of the cortical tissue or adjacent to it inthe surrounding tissue. At sufficiently high concentrations, the silvercation will be fully biocidal. Thus, silver ranging from 10 to 10,000parts per million may be used.

It is also envisioned to add soluble silver to the surface aftertreatment to provide biostatic properties inhibiting any growth ofmicroorganisms which may be resident on the surface of the corticaltissue or adjacent to it in the surrounding tissue. Silver which can beadded is can be taken from a group consisting of silver nitrate andother soluble or slightly soluble silver compounds such as silverchloride, silver oxide, silver sulphate, silver phosphate, silveracetate, silver perchlorate or silver tartrate.

It is also possible to add one or more rhBMP's to the surface of thetreated bone shape by soaking and being able to use a significantlylower concentration of the rare and expensive recombinant human BMP toachieve the same acceleration of biointegration. The addition of otheruseful treatment agents such as vitamins, hormones, antibiotics,antiviral and other therapeutic agents could also be added to thesurface modified layer. BMP directs the differentiation ofpluripotential mesenchymal cells into osteoprogenitor cells which formosteoblasts. The ability of freeze dried demineralized cortical bone tofacilitate this bone induction principle using BMP present in the boneis well known in the art. However, the amount of BMP varies in the bonedepending on the age of the bone donor and the bone processing.Sterilization is an additional problem in processing human bone formedical use as boiling, autoclaving or irradiation over 2.0 Mrads issufficient to destroy or alter the BMP present in the bone matrix.

The time, temperature and acid concentration can be adjusted to achievea set of process conditions that will give the same physical result asthe above noted examples. Temperature could be lowered to 4° C. andallow the process time to increase to one hour (a four fold increase inprocess time). Temperatures much above 30° C. will result in too rapid arate of hydroxyapatite removal and result in a highly variable shape.Conditions could be adjusted to use acid concentrations from about 0.1Nto about 2.0N HCl. Lower concentrations will result in a very slow rateof mineral layer removal, not conducive to a commercial process. Higherconcentrations will result in a too rapid rate of mineral removal and toa highly varied and uncontrolled surface. Other acids could be used;sulfuric, phosphoric or other mineral acids, organic acids such asacetic; chelating agents such as ethylene diamine tetra acetic acid orother weak acids would also be suitable.

Any number of medically usefull substances can be incorporated in theinvention by adding the substances to the composition at any steps inthe mixing process or directly to the final composition. Such substancesinclude collagen and insoluble collagen derivatives, hydroxyapatite andsoluble solids and/or liquids dissolved therein. Also included areantiviricides such as those effective against HIV and hepatitis;antimicrobial and/or antibiotics such as erythromycin, bacitracin,neomycin, penicillin, polymyxin B, tetracycline, viomycin, chloromycetinand streptomycin, cefazolin, ampicillin, azactam, tobramycin,clindamycin and gentamycin. It is also envisioned that amino acids,peptides, vitamins, co-factors for protein synthesis; hormones;endocrine tissue or tissue fragments; synthesizers; enzymes such ascollagenase, peptidases, oxidases; polymer cell scaffolds withparenchymal cells; angiogenic drugs and polymeric carriers containingsuch drugs; collagen lattices; biocompatible surface active agents,antigenic agents; cytoskeletal agents; cartilage fragments, living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells, naturalextracts, tissue transplants, bioadhesives, transforming growth factor(TGF-beta), insulin-like growth factor (IGF-1); growth hormones such assomatotropin; bone digestors; antitumor agents; fibronectin; cellularattractants and attachment agents; immuno-suppressants; permeationenhancers, e.g. fatty acid esters such as laureate, myristate andstearate monoesters of polyethylene glycol, enamine derivatives,alpha-keto aldehydes can be added to the composition.

All products can also be done in an aseptic environment to maintain asterile final product or sterilized after production. The cortical bonestructure is then placed in a moisture permeable inner container whichis placed in a moisture barrier outer container.

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.However, the invention should not be construed as limited to theparticular embodiments which have been described above. Instead, theembodiments described here should be regarded as illustrative ratherthan restrictive. Variations and changes may be made by others withoutdeparting from the scope of the present invention as defined by thefollowing claims.

What we claim is:
 1. A sterile bone structure for application to a bonedefect site to promote new bone growth at the site comprising a singleallograft bone body with an outer partially demineralized cortical bonesection and a central mineral bone core section, said bone structureafter the demineralization process retaining a cross sectional surfacearea ranging from about 85% to about 95% of the original mineralizedbone surface area before demineralization with the remaining partiallydemineralized cortical bone structure comprising an outer demineralizedlayer ranging from about 0.05 mm to about 0.08 mm in thickness.
 2. Asterile bone structure as claimed in claim 1 wherein said bone structureincludes bone morphogenic proteins in excess of the amount naturallyoccurring in allogenic bone.
 3. A sterile bone structure as claimed inclaim 1 wherein said structure is a pin.
 4. A sterile bone structure asclaimed in claim 1 wherein said structure is a plate.
 5. A sterile bonestructure as claimed in claim 1 wherein said structure is a screw.
 6. Asterile bone structure as claimed in claim 1 wherein said structure is arod.
 7. A sterile bone structure as claimed in claim 1 wherein saidstructure is a wedge.
 8. A sterile bone structure as claimed in claim 1wherein said structure is a rod.
 9. A sterile bone structure as claimedin claim 1 wherein said structure is an anchor.
 10. A sterile bonestructure as claimed in claim 1 wherein said structure is a fusion ring.11. A sterile bone structure as claimed in claim 1 wherein saidstructure is a fusion block.
 12. A sterile malleable bone composition asclaimed in claim 1 including antimicrobial and/or antibiotics selectedfrom the group consisting of erythromycin, bacitracin, neomycin,penicillin, polymyxin B, tetracycline, viomycin, chloromycetin andstreptomycin, cefazolin, ampicillin, azactam, tobramycin, clindamycinand gentamycin added to the demineralized layer of said bone structure.13. A sterile bone structure as claimed in claim 1 including a solublesilver compound added to said demineralized layer of said bonestructure.
 14. A sterile bone structure as claimed in claim 13 whereinsaid soluble silver compound contains silver in a range of 10 to 10,000parts per million.
 15. A sterile bone structure as claimed in claim 13wherein said silver compound is taken from a group consisting of silvernitrate, silver chloride, silver oxide, silver sulphate, silverphosphate, silver acetate, silver perchlorate, or silver tartrate.
 16. Asterile bone structure for application to a bone defect site to promotenew bone growth at the site comprising a partially demineralizedcortical bone structure with a central mineral bone section, said bonestructure after the demineralization process retaining a cross sectionalsurface area ranging from about 85% to about 95% of the originalmineralized bone surface area before demineralization with the remainingpartially demineralized cortical bone structure comprising an outerdemineralized portion ranging from about 5% to about 15% of the crosssectional area of the demineralized cortical bone structure.
 17. Asterile partially demineralized bone structure for application to a bonedefect site to promote new bone growth at the site comprising apartially demineralized single piece cortical bone structure with athickness in excess of 2 mm, said cortical bone structure being providedwith an outer surface layer of demineralized bone having a thicknessranging from about 0.05 mm to about 0.11 mm and a central mineralizedsection.
 18. A sterile partially demineralized bone structure forapplication to a bone defect site to promote new bone growth at the sitecomprising a single piece partially demineralized cortical bonestructure with a thickness in excess of 2 mm, said bone structure havingan outer surface layer of demineralized bone with a thickness rangingfrom about 0.14 mm to about 0.17 mm and a central mineralized sectionhaving a cross sectional area of at least 2 times the cross sectionalarea of said demineralized layer.
 19. A sterile partially demineralizedbone structure for application to a bone defect site to promote new bonegrowth at the site comprising a unitary cortical formed bone device witha partially demineralized cortical bone structure with a thickness inexcess of 2 mm having an outer surface layer of demineralized bonehaving a thickness ranging from about 0.08 mm to about 0.11 mm and acentral mineralized section having a cross sectional area of at least 3times the cross sectional area of said demineralized layer.